Project Summary This project will overcome a main obstacle impeding progress to fully exploit marine natural products (NPs) for health applications. Currently, there are no efficient genetic methods to interrogate and modify endogenous NP biosynthetic pathways from marine filamentous cyanobacteria, and no robust platforms for heterologous expression and genetic engineering of cyanobacterial NP pathways. This project will develop methods and tools for engineering cyanobacteria for the heterologous expression and manipulation of NP gene clusters identified in organisms that are not amenable to genetic methods, or orphan NP gene clusters that are identified in environmental DNA sequences. The project will use the synthetic-biology strain Synechococcus elongatus PCC7942 and the marine filamentous cyanobacterium Leptolyngbya (ISB-3N94-8PLP) to provide two distinct but complementary genetic platforms for the expression and engineering of NP pathways. The long-term objectives of this project are to provide efficient platforms for the production of NPs in quantities suitable for studying their biological activities and for chemical modifications to enhance those activities for health applications. Although a few relatively simple marine cyanobacterial NPs have been produced in heterologous hosts such as E. coli, difficulties remain for the expression certain enzymes and of large complex pathways in phylogenetically distant hosts. We hypothesize that expression of these cyanobacterial genes and gene clusters in cyanobacterial hosts will overcome this obstacle. The 3 specific aims of this research are as follows. (1) Establish Leptolyngbya as a broadly applicable genetic platform for identification, expression, and interrogation of NP pathways. (2) Develop improved genetic tools to enable the transfer, refactoring, and overexpression of large biosynthetic pathways in platform strains of cyanobacteria. (3) Express two orphan pathways from the marine cyanobacterial genus Okeania in S. elongatus and Leptolyngbya. New genetic tools and methods will be created for S. elongatus and Leptolyngbya that will include TAR cloning vectors, shuttle plasmids, chromosomal integration sites, constitutive and regulated promoters, reporter genes, and antibiotic-resistance markers to facilitate manipulation of endogenous NP pathways and for the heterologous expression of pathways from other organisms. This project will combine state-of-the-art approaches in genomics, transcriptomics, bioinformatics of secondary metabolite pathways, cyanobacterial genetic engineering, and NP chemistry to address knowledge gaps related to medically important NP biosynthesis in marine cyanobacteria. The project will develop Leptolyngbya and S. elongatus into broadly useful expression hosts for cyanobacterial secondary-metabolite enzymes and entire NP biosynthetic pathways, and these hosts and genetic tools will be made available to the NP research community.